Colorectal cancer is the commonest internal malignancy affecting men and women in Australia. About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990. In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.
Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer. Incidence rates vary approximately 20-fold around the world, with the highest rates seen in the developed world and lowest in India. Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand. Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.
Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body. Survival rates are significantly improved where the disease is detected and treated early.
The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors. Recognition of the genetic component of colorectal cancer is growing. Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult. There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer. FAP and HNPCC contribute to approximately 1% and 4%, respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10-15% of patients.
However, in the vast majority of patients the aetiology of large bowel cancer remains unknown. Most colon cancer arises within pre-existing benign precursor lesions or adenomas. Adenomas are classified by histological architecture as tubular, tubulovillous or villous. Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia. Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.
Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers. For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp. Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise. The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes. This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.
Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites. Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels. It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases. Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers. These events are thought to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.
Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.) In general, these cell surface molecules mediate both cell-cell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin. It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.
Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins. Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus internalisation and tumourigenesis. Integrins are transmembrane glycoproteins consisting of an alpha (α) and beta (β) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell. The integrin family comprises 17 different α and 8 β subunits and the αβ combinations are subsumed under 3 subfamilies.
Excluding the leucocyte integrin subfamily that is designated by the β2 nomenclature, the remaining integrins are arranged into two major subgroups, designated β1 and αv based on sharing common chains.
In the β1 subfamily, the β1 chain combines with any one of nine α chain members (α1-9), and the a chain which associates with β1 determines the matrix-binding specificity of that receptor. For example, α2β1 binds collagen and laminin, α3β1 binds collagen, laminin and fibronectin, and α5β1 binds fibronectin. In the αv subfamily of receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).
The current picture of integrins is that the N-terminal domains of α and β subunits combine to form a ligand-binding head on each integrin. This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains. The β subunits all show considerable similarity at the amino acid level (Loftus et al, 1994). All have a molecular mass between 90 and 110 kDa, with the exception of β4 which is larger at 210 kDa. Similarly, they all contain 56 conserved cysteine residues, except for β4 which has 48. These cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds. The α-subunits have a molecular mass ranging from 150-200 kDa. They exhibit a lower degree of similarity than the β chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.
The β subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992). In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include α-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992). At least three different regions that are required for focal contact localisation of β1 integrins have been delineated (Reszka et al, 1992). These regions contain conserved sequences that are also found in the cytoplasmic domains of the β2, β3, β5, β6 and β7 integrin subunits. The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.
Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, increases in intracellular Ca2+ levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes. In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995). Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.
The integrin β6 subunit was first identified in cultured epithelial cells as part of the αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992). The β6 subunit is composed of 788 amino acids and shares 34-51% sequence homology with other β integrin subunits β1-β5. The β6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990). The cytoplasmic domain differs from other β subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin β subunits, and a unique 11 amino acid carboxy-terminal extension. The 11 amino acid extension has been shown not to be necessary for localisation of β6 to focal contacts; in fact, its removal appears to increase receptor localisation. However, removal of any of the three conserved regions previously identified as important for the localisation of β1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of β6 to focal contacts (Cone et al, 1994).
The integrin αvβ6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo, and this growth-enhancing effect is due, at least in part, to αvβ6-mediated gelatinase B secretion (Agrez et al, 1994; Niu et al, 1998 Agrez et al, 1999). What makes this epithelial-restricted integrin of particular interest in cancer is that it is not expressed on normal epithelial cells but is highly expressed during would healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996). Further more, αvβ6 has been found to induce its own expression in an autocrine manner with cell crowding and a self-perpetuating model of colon cancer progression regulated by αvβ6-mediated gelatinase B secretion has been proposed (Niu et al, 2000).
Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs). The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear. However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).
Integrins can signal through the cell membrane in either direction. The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999). Outside-in signalling can be roughly divided into two descriptive categories. The first is ‘direct signalling’ in which ligation and clustering of integrins is the only extracellular stimulus. Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases (e.g. focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4,5-biphosphate (P1P2) synthesis. The second category of integrin signalling is ‘collaborative signalling’, in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998). In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.
MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane. The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinases (e.g. Raf-1) (Garrington and Johnson, 1999). Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms. ERK 1/2 (44 kD and 42 kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991). These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90rsk) cytoskeletal proteins (such as microtubule-associated phospholipase A2), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1). ERKs play a major role in growth-promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and Ruoslahti, 1999).
MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases. The FAK pathway is activated by most integrins. In addition to activating FAK, some β1 and αv integrins also activate the tyrosine kinase Fynu and through it, the adaptor protein Shc (Wang et al, 1996). It is likely that both FAK and Shc contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997). It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997). The laminin receptor α6β4, the laminin/collagen receptor α1β1, the fibronectin receptor α5β1 and the RGD binding receptor αvβ3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997). The ability of integrins to activate ERK may be especially important when the concentration of growth factors available to the cell is limited. In this setting, proliferation is likely to require co-stimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999). Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrix-degrading enzyme secretion.
While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein Shc (Howe et al, 1998; Giancotti & Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs. For example, MEK-independent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).
Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of Shc. For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999). Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.
The importance of the MAP kinase pathway in promoting colon cancer growth in vivo is now no longer in question. Although relatively little is known about the role of MAP kinase activation in for instance, colon tumour progression, MAP kinase pathways have been shown to be highly activated during the late progression of colorectal cancer (Licato et al, 1997). In a recent breakthrough in this field, a highly potent inhibitor of MAP kinase activation has been identified which is capable of inhibiting human colon cancer growth in immune-deficient mice raising hops for the clinical application of MAP kinase inhibitors in the treatment of colon cancer (Sebolt-Leopold et at, 1999).
Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains. Direct interactions have been identified between cytoskeletal proteins such as α-actinin and talin and β1 and β3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998). A direct association between FAK and the β1 integrin tail has been suggested based on in vitro β1 peptide studies, but this remains to be confirmed (Schaller et al, 1995). More recently, the cytoplasmic domain of the α4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).
Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate “inside-out” integrin signalling. For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify α2β1 integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995). Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the β1, β2 and β3 subunits. When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996). Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans). The extracellular Ig region of the IAP molecule mediates association with αvβ3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996). An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinositol-4-kinase and PKC into complexes with integrins (Hemler, 1998).
Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors. For example, the integrin αvβ6 has been shown to bind and activate latent TGFβ1 in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury. In melanoma cells, αvβ3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997). Synergism between integrin-mediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF β-receptor is tyrosine phosphorylated upon growth factor stimulation. Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the αvβ3 ligand, vitronectin.